This proposal will enable the principal investigator to develop into an independent physician-scientist and gain new expertise in oxidative biology, gene regulation, and cystic fibrosis (CF). CF is a common, highly morbid, and ultimately fatal disease characterized by persistent pseudomonas lung infections and severe airways obstruction. It is also associated with markedly elevated concentrations of iron in the airway that may contribute to CF morbidity by generating oxidant injury, and supplying pseudomonas with an essential nutrient to exacerbate infection. Airway epithelial cells protect against these effects by transporting iron via natural resistance-associated macrophage protein 2 (Nramp-2) into the cell where it is sequestered by ferritin. To prevent intracellular accumulation, iron is mobilized from lung cells via metal transport protein 1 (MTP1). We present data to support two potential mechanisms of intracellular iron accumulation in CF. First, inflammation appears to increase Nramp-2 expression, decrease MTP1 expression, and elevate intracellular iron concentration. Second, basal expression of Nramp-2 and MTP1 appears elevated in unstimulated CF cells. This may be due to increased Na+ absorption via the epithelial channel (ENaC), that is typical in CF, given our data showing iron transport requires concurrent Na+ transport and ferritin is increased in airways of mice over-expressing ENaC. We will test the hypotheses that: 1) Inflammation increases Nramp-2 expression and decreases MTP1 expression in airway epithelial cells resulting in intracellular iron overload;2) Increased intracellular iron, in the presence of inflammation, causes ROS generation and oxidant injury;and 3) In addition to and independently of inflammation, Nramp-2 and MTP1 expression are mediated by increased expression of ENaC in CF epithelial cells. Three Specific Aims will be tested: 1) Evaluate the extent to which LPS and proinflammatory cytokines IL-1B and TNF-a regulate iron transport proteins and increase cellular iron concentration in airway epithelial cells;2) Evaluate whether elevated intracellular iron during inflammation stimulates ROS production and generates oxidative injury;3) Investigate the role of increased ENaC expression in the regulation of Nramp-2 and MTP1 expression in CF in the presence and absence of inflammatory stimuli. These data will provide a scientific foundation to understand mechanisms of iron overload in CF and its role in the vulnerability of the airway to oxidative damage and cytotoxicity. (End of Abstract)